Previous work has established that the melanocyte-specific tyrosinase-related protein-1 (TRP-1) promoter is regulated positively by the microphthalmia-associated transcription factor Mitf, acting through the conserved M box and negatively by the T-box factor Tbx2, which can bind two "melanocyte-specific elements" termed the MSEu and MSEi. Both the MSEu and MSEi, which share a 6-base pair GTGTGA consensus, are also recognized by a previously unidentified melanocyte-specific factor, MSF. Here we show using a combination of DNA binding assays, proteolytic clipping, and anti-Pax3 antibodies that MSF is indistinguishable from Pax3, a paired homeodomain transcription factor implicated genetically in melanocyte development and the regulation of the Mitf promoter. Consistent with Pax3 being able to bind the TRP-1 promoter, Pax3 is expressed in melanocytes and melanomas, and TRP-1 promoter activity is up-regulated by Pax3. The results identify a novel role for Pax3 in the expression of TRP-1, and the potential role of Pax3 in the melanocyte lineage is discussed.